Abstract:
:MicroRNAs are small regulatory molecules that demonstrate useful biomarker potential. They have been recognised in biofluids, where they are protected from degradation by encapsulation into extracellular vesicles (EVs). A number of commercial products are available for the isolation of EVs and their RNA content; however, extensive protocol comparisons are lacking. Furthermore, robust qRT-PCR assessment of microRNA expression within EVs is problematic, as endogenous controls (ECs) previously used in cellular samples may not be present. This study compares EV isolation and RNA extraction methods (EV precipitation reagents, RNA isolation kits and ultracentrifugation) from serum or urine samples and evaluates suitable ECs for incorporation into qRT-PCR analysis. Results were assessed by electron microscopy, nanoparticle tracking analysis and bioanalyzer concentrations. The stability of 8 ECs was compared for both serum and urine EV RNA and retrospectively validated in independent cohorts (serum n=55, urine n=50). The Life Technologies precipitation reagent gave superior serum EV recovery compared to SBI reagent, as assessed by NTA size distribution, increased RNA concentration, and lower small RNA Ct values. Similarly, the Norgen Biotek Urine Exosome RNA Isolation Kit gave improved results for urine EV isolation compared to ultracentrifugation, when determined by the same parameters. The Qiagen miRNeasy™ RNA isolation kit gave suitable serum EV RNA concentrations compared to other kits, as assessed by Bioanalyzer and small RNA qRT-PCR. Small RNAs HY3 (S.D=1.77, CoV=6.2%) and U6 (S.D=2.14, CoV=8.6%) were selected as optimal ECs for serum EV microRNA expression analysis, while HY3 (S.D=1.67, CoV=6.5%) and RNU48 (S.D=1.85, CoV=5.3%) were identified as suitable for urine studies. In conclusion, this study identifies optimal methods for isolation of serum and urine EV RNA, and suitable ECs for normalisation of qRT-PCR studies. Such reports should aid in the standardisation of EV microRNA data, particularly for biomarker studies.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Crossland RE,Norden J,Bibby LA,Davis J,Dickinson AMdoi
10.1016/j.jim.2015.12.011subject
Has Abstractpub_date
2016-02-01 00:00:00pages
39-49eissn
0022-1759issn
1872-7905pii
S0022-1759(15)30077-6journal_volume
429pub_type
杂志文章abstract::One major obstacle in antibody production is the lack of highly purified immunogen. In this study, we describe an alternative strategy to circumvent this problem. A nucleotide sequence encoding a full-length of human survivin was cloned into pAK400cb. After transforming into an E. coli Origami B strain, survivin-bioti...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2006.07.024
更新日期:2006-12-20 00:00:00
abstract::An ELISA method for the detection of monoclonal antibodies (MAb) to the T3-T cell antigen receptor (TCR) complex was devised. The T3-TCR complex was solubilised using digitonin and a rat anti-T3 MAb (Campath 3) was used to bind it to an ELISA plate. Normal rat serum was used to block cross-reactivity between the rat M...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(88)90280-3
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(89)90115-4
更新日期:1989-02-08 00:00:00
abstract::Utilization of compounds that enhance the innate immune response against Mycobacterium tuberculosis is an attractive strategy for combating tuberculosis in the post-antibiotic era. Thus, it is crucial to develop methods that can be used to screen for such compounds and to investigate their mechanisms of action. Here, ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2015.10.003
更新日期:2015-12-01 00:00:00
abstract::The attachment of cells to plastic tissue culture plates is a widely used criterion of cell viability in microcytotoxicity assays. When cell suspensions of primary human colon carcinoma and melanoma cells which were of low initial viability (assessed by trypan blue exclusion) were allowed to adhere to tissue culture p...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(81)90039-9
更新日期:1981-01-01 00:00:00
abstract::We have discovered that 0.5-0.8 M ammonium sulfate has a beneficial effect on the peptic digestion of several antibodies. Initially, the project was begun to address the troublesome precipitation observed when pepsin was used to digest monoclonal antibody CYT-368 (C46), an IgG2a that binds carcinoembryonic antigen. Hi...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(93)90083-j
更新日期:1993-01-04 00:00:00
abstract::With the use of a panel of monoclonal antibodies against structurally related carbohydrate antigens, finely regulated cellular phenotype of the carbohydrate antigens can be characterized. As a model system, the expression of N-acetyllactosamine-related antigens in human biliary epithelial cells was investigated using ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(88)90364-x
更新日期:1988-09-13 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(12)80050-0
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(99)00012-5
更新日期:1999-04-22 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(99)00240-9
更新日期:2000-04-03 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(88)90414-0
更新日期:1988-04-06 00:00:00
abstract::The cell mediated immune response to herpes simplex virus was assessed by a modification of the leukocyte migration inhibition test, developed to increase its sensitivity and correlated with the in vitro stimulation of lymphocytes. The introduction of a preincubation step allowed us to demonstrate significant inhibiti...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(76)90202-7
更新日期:1976-01-01 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2017.03.008
更新日期:2017-06-01 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(77)90058-8
更新日期:1977-01-01 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(88)90176-7
更新日期:1988-11-10 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(97)00121-x
更新日期:1997-08-22 00:00:00
abstract::It is important to know the contribution of specific allergens to a complex allergenic extract and to have a dependable method to assess the effector activity of an extract specifically depleted of that allergen. We have previously shown that removal of the major peanut allergen, Ara h 2, from a crude peanut extract (...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2011.06.031
更新日期:2011-09-30 00:00:00
abstract::Macrophages are an important part of the cellular immune system and play a key role during immune responses. Thus, macrophages are interesting targets in basic and clinical research. Primary monocytes or monocyte-derived macrophages do not proliferate on a suitable scale so that their use for functional studies in vit...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2009.03.014
更新日期:2009-05-31 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(77)90130-2
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(82)90322-2
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(90)90044-v
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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更新日期:1991-01-24 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(83)90428-3
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2014.03.021
更新日期:2014-05-01 00:00:00
abstract::The study of T cell receptor (TCR) genes has been hampered by their large repertoires and elusive methods for gene amplification. We have developed a new method for amplification of all human TCR genes (alpha, beta, gamma, and delta) with the ligation of a universal adaptor to the leader sequence of variable (V) regio...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(93)90193-b
更新日期:1993-05-05 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(97)00186-5
更新日期:1997-12-29 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(87)90363-2
更新日期:1987-01-26 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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更新日期:2000-01-13 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(86)90338-8
更新日期:1986-02-27 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(80)90042-3
更新日期:1980-01-01 00:00:00